Artemisinin (AN), a sesquiterpene lactone, and its derivatives are proven, effective antimalarial therapeutics that also show anticancer activity. However, the complex compound is still best produced in plants; and the drug is in short supply. AN can be produced in transformed root cultures of Artemisa annua L.; we have considerable experience with controlling production. Since to our knowledge it has not yet been established which of the two arms of the terpenoid biosynthetic pathway are operational in the biosynthesis of artemisinin, we hypothesize that AN is produced from a pool of IPP that originates from both the mevalonate and the non-mevalonate pathway, as shown for sesquiterpenes of chamomile, a close relative of Artemisia annua L. Since the sesquiterpene cyclase, ADS, leads to AN and has recently been, we hypothesize that this gene is also present and responsive in our transformed roots that are known producers of artemisinin. Objectives: to be accomplished over 2.5 years (% effort): 1. To do C13 labeling experiments to determine the fate of carbon in artemisinin biosynthesis so that we might know whether one, the other, or both arms of the terpenoid biosynthetic pathway are playing a role in its production. (65%) 2. To produce a molecular probe for the specific sesquiterpene cyclase (SQC) gene, amorpha-4,11-diene synthase (ADS), that leads to artemisinin in order to study any future ADS expression in hairy roots if roots ire ever to be considered as a production system for artemisinin. (15%) 3. To demonstrate that expression of the ADS probe correlates with artemisinin production using conditions where production is known to be either very high or very low. (20%) The long-term goal of our work is to improve fundamental understanding of the regulatory control of terpene production in hairy roots so that we can demonstrate their utility as a production system for important secondary metabolites. This study will enhance our understanding of the biosynthesis of AN in plants and further our ability to increase production of artemisinin while improving overall understanding of terpenoid biosynthesis.